Polymer-von Willebrand factor-conjugates

ABSTRACT

The present invention relates to a proteinaceous construct (also designated as polymer-VWF-conjugate) comprising plasmatic and/or recombinant von Willebrand factor (VWF), said VWF being bound to at least one physiologically acceptable polymer molecule, as well as to a complex between said proteinaceous construct and at least one factor VIII (FVIII) protein. The physiologically acceptable polymer molecule can be, for instance, polyethylene glycol (PEG) or polysialic acid (PSA). Further the present invention relates to methods for prolonging the in vivo-half-life of VWF or FVIII in the blood of a mammal having a bleeding disorder associated with functional defects of or deficiencies of at least one of FVIII or VWF.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationser. No. 11/317,582, filed Dec. 23, 2005, which claims priority to U.S.Patent Application Ser. No. 60/639,244, filed Dec. 27, 2004, U.S. PatentApplication Ser. No. 60/668,378, filed Apr. 4, 2005, U.S. PatentApplication Ser. No. 60/671,901, filed Apr. 15, 2005, and U.S. PatentApplication Ser. No. 60/685,086, filed May 26, 2005, the entirerespective disclosures of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a proteinaceous construct comprisingplasmatic and/or recombinant von Willebrand factor (VWF), said VWF beingbound to at least one physiologically acceptable polymer molecule, aswell as to a complex between said proteinaceous construct and at leastone factor VIII (FVIII) protein. Further the present invention relatesto methods for prolonging the in vivo half-life of VWF or FVIII in theblood of a mammal having a bleeding disorder associated with functionaldefects or deficiencies of at least one of FVIII or VWF.

BACKGROUND OF THE INVENTION

VWF is a multimeric adhesive glycoprotein present in the plasma ofmammals, which has multiple physiological functions. During primaryhemostasis VWF acts as a mediator between specific receptors on theplatelet surface and components of the extracellular matrix such ascollagen. Moreover, VWF serves as a carrier and stabilizing protein forprocoagulant FVIII. VWF is synthesized in endothelial cells andmegakaryocytes as a 2813 amino acid precursor molecule. The precursorpolypeptide, pre-pro-VWF, consists of a 22-residue signal peptide, a741-residue pro-peptide and the 2050-residue polypeptide found in matureplasma VWF (Fischer et al., FEBS Lett. 351: 345-348, 1994). Uponsecretion into plasma VWF circulates in the form of various species withdifferent molecular sizes. These VWF molecules consist of oligo- andmultimers of the mature subunit of 2050 amino acid residues. VWF can beusually found in plasma as one dimer up to multimers consisting of50-100 dimers (Ruggeri et al. Thromb. Haemost. 82: 576-584, 1999). Thein vivo half-life of human VWF in the human circulation is approximately12 to 20 hours.

The most frequent inherited bleeding disorder in humans is vonWillebrand's disease (VWD), which can be treated by replacement therapywith VWF containing concentrates of plasmatic or recombinant origin. Dueto the short half-life of VWF in blood there is a strong need to developVWF concentrates with a prolonged in vivo half-life of VWF. The sameapplies to FVIII, which has also a relatively short in vivo half-life ofapproximately 8 to 12 hours requiring frequent re-dosing for patienttreatment of bleeding disorders associated with functional defects of ordeficiencies of at least one of FVIII and VWF.

In the prior art it has been described that recombinant VWF (rVWF)produced in an eucaryotic cell culture is more intact and lessproteolytically degraded than plasma-derived VWF (Fischer et al., FEBSLett. 375: 259-262, 1995). EP 0 784 632 describes a method for isolatinghighly pure VWF by purifying recombinant VWF using anion exchangechromatography. Methods for a large scale production of homogenous andstructurally intact VWF are also known in the art (Schlokat et al.,Biotechnol. Appl. Biochem. 24: 257-267, 1996; Fischer et al., CMLS 53:943-950, 1997). Recombinant VWF has been characterized by using canine,murine, and porcine models of von Willebrand's disease (VWD) (Turecek etal., Blood 90: 3555-3567, 1997; Roussi et al., Blood Coag. Fibrinol. 9:361-372, 1998; Schwarz et al., Haemophilia 4: 53-62, 1998; Schwarz etal., Semin. Thromb. Hemost. 28: 215-225, 2002). WO 00/49047 describes amethod for producing a VWF preparation by treating pro-VWF withthrombin. A method for purifying proteins that bind to VWF by using arVWF immobilized on a carrier is disclosed in WO 98/25969. Thepharmaceutical use of plasma derived and recombinant VWF pro-peptides(pro-VWF) for treating blood coagulation disorders is described in EP 0977 584. U.S. Pat. No. 6,037,452 describes the binding of FVIII andfactor IX (FIX) to a poly(alkylene oxide) through a linker or a couplingagent. In EP 0 774 261 it is shown that the use of recombinant VWFhaving a prolonged biological in vivo half-life stabilizes FVIII in theblood of a mammal and induces the production of endogenous FVIII.Nevertheless, there exists a need for patients having VWF- orFVIII-based bleeding disorders to further increase the in vivo half-lifeof VWF and FVIII.

VWF is known to stabilize FVIII in vivo and, thus, plays a crucial roleto regulate plasma levels of FVIII and as a consequence is a centralfactor to control primary and secondary hemostasis. It is also knownthat after application of therapeutic products containing VWF anincrease in endogenous FVIII:C to 1 to 3 units per ml in 24 hours can beobserved demonstrating the in vivo stabilizing effect of VWF on FVIII.

A strong need exists for a new substance for widening the treatmentspectrum for deficiencies in coagulation FVIII also known as hemophiliaA and/or qualitative or quantitative deficiencies of VWF also known asVWD. Due to a lack of functional VWF, patients with VWD have a secondarydefect of FVIII represented by FVIII plasma levels below normal.Depending on the type of VWD and the severity of the diseases theseFVIII levels can vary but are generally measurably lower than the FVIIIplasma level found in healthy humans.

Thus, the present invention provides a novel system for prolonging thein vivo half-life of VWF and/or of FVIII in the blood of a mammal. It isa further object of the present invention to provide methods for theimproved treatment of bleeding disorders associated with functionaldefects of or deficiencies of one or both of FVIII and VWF.

SUMMARY OF THE INVENTION

The present invention relates to a proteinaceous construct comprisingplasmatic and/or recombinant von Willebrand factor (VWF) or biologicallyactive derivatives thereof, said VWF or said biologically activederivatives thereof being bound to one or more physiologicallyacceptable polymer molecules, wherein the in vivo half-life of theproteinaceous construct is prolonged in the blood of a mammal,particularly a human. Further, the present invention relates to acomplex between said proteinaceous construct and at least one factorFVIII (FVIII) protein or a biologically active derivative thereof,wherein the in vivo half-life of said FVIII protein or said biologicallyactive derivative thereof is also prolonged in the blood of a mammal.Additionally, pharmaceutical compositions containing said proteinaceousconstruct or said complex as well as methods for prolonging the in vivohalf-life of VWF or FVIII in the blood of a mammal having a bleedingdisorder associated with functional defects of or deficiencies of atleast one of FVIII and VWF using said proteinaceous construct or saidcomplex are provided according to the present invention. Methods formaking the proteinaceous construct are also provided.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention relates to a proteinaceous construct(in the following also designated as “polymer-VWF-conjugate”) comprisingplasmatic and/or recombinant VWF or biologically active derivativesthereof, said VWF or said biologically active derivatives thereof beingbound to one or more types of physiologically acceptable polymermolecules, wherein the in vivo half-life of said VWF or saidbiologically active derivatives thereof is prolonged in the blood of amammal.

It is a further aspect of the present invention to providepolymer-VWF-conjugates, which follow two principal pharmacologicalmechanisms and of forms of said polymer-VWF-conjugates having featuresin-between said two forms. One form stably carries the polymerconjugated to VWF and will be eliminated as an integral molecule overtime after application to a mammal. The other form is characterized byreversibility of the polymer conjugated to VWF. After administration toa mammal, the polymer molecules bound to VWF will be gradually releasedfrom VWF and non-conjugated VWF will become available as thepharmacologically functional agent. The release characteristics willdepend on the conjugation chemistry and on the composition and thestructure of the polymer molecules bound to VWF.

The polymer-VWF conjugates are useful either alone for treatment of VWD,or combined with FVIII to stabilize FVIII for increased half-life orboth. When used solely for the treatment of VWD, the conjugate may takeone of two forms. The first form is where the polymer is releasablybound to the VWF. In this manner the VWF becomes active as the polymeris released or degrades. The second form is where the polymerconcentration bound to the VWF is such so as to not interfere with theVWF activity. When the conjugate is prepared to bind with and stabilizeFVIII, the degree or level of polymer bound to the VWF is provided so asto not interfere with the binding region of the VWF. As will be shown inthe examples, satisfactory polymer conjugation to the VWF can beachieved without interfering with the VWF and FVIII binding capacity.The degree of polymer conjugation can also be controlled or modified tomaintain VWF activity while also maintaining the ability of the VWF tobind FVIII. In this form the polymer-VWF conjugates provide atherapeutically active VWF, while also stabilizing FVIII for increasedhalf-life.

The VWF and FVIII molecules useful for the present invention include thefull length protein, precursors of the protein, subunits or fragments ofthe protein, and functional derivatives thereof. Reference to VWF andFVIII or FVIII is meant to include all potential forms of such proteins.

As used herein “biologically active derivative” includes any derivativeof a molecule having substantially the same functional and/or biologicalproperties of said molecule, such as binding properties, and/or the samestructural basis, such as a peptidic backbone or a basic polymeric unit.

The VWF useful for the present invention includes all potential forms,including the monomeric and multimeric forms. One particularly usefulform of VWF are homo-multimers of at least two VWFs. The VWF proteinsmay be either a biologically active derivative, or when to be usedsolely as a stabilizer for FVIII the VWF may be of a form notbiologically active. It should also be understood that the presentinvention encompasses different forms of VWF to be used in combination.For example, a composition useful for the present invention may includedifferent multimers, different derivatives and both biologically activederivatives and derivatives not biologically active. In primaryhemostasis VWF serves as a bridge between platelets and specificcomponents of the extracellular matrix, such as collagen. The biologicalactivity of VWF in this process can be measured by two different invitro assays (Turecek et al., Semin. Thromb. Hemost. 28: 149-160, 2002).The ristocetin cofactor assay is based on the agglutination of fresh orformalin-fixed platelets induced by the antibiotic ristocetin in thepresence of VWF. The degree of platelet agglutination depends on the VWFconcentration and can be measured by the turbidimetric method, e.g. byuse of an aggregometer (Weiss et al., J. Clin. Invest. 52: 2708-2716,1973; Macfarlane et al., Thromb. Diath. Haemorrh. 34: 306-308, 1975).The second method is the collagen binding assay, which is based on ELISAtechnology (Brown et Bosak, Thromb. Res. 43: 303-311, 1986; Favaloro,Thromb. Haemost. 83: 127-135, 2000). A microtiter plate is coated withtype I or III collagen. Then the VWF is bound to the collagen surfaceand subsequently detected with an enzyme-labeled polyclonal antibody.The last step is the substrate reaction, which can be photometricallymonitored with an ELISA reader.

As used herein, “plasma-derived VWF (pdVWF)” includes all forms of theprotein found in blood including the mature VWF obtained from a mammalhaving the property of in vivo-stabilizing, e.g. binding, of at leastone FVIII molecule. However, the invention is not limited to the matureVWF. One, biologically active derivative of said pVWF is pro-VWF whichcontains the pro-peptide. Other forms of VWF useful for the presentinvention include the proteinaceous construct comprises immature VWFincluding the precursor VWF molecule (pre-pro-VWF) synthesized byendothelial cells and megakaryocytes, and/or the VWF propeptide(pro-VWF) and/or mature pdVWF obtained upon cleavage of the signalpeptide and pro-peptide, respectively of the precursor molecule. Furtherexamples of biologically active derivatives of pdVWF include pro-drugswhich are processed or converted into the biologically active form, oris biologically active as such, truncated forms, forms having deletions,forms having substitutions, forms having additions other than pro-forms,fragments of the mature form, chimeric forms, and forms havingpost-translational modifications as compared to the natural form. PdVWFuseful for the present invention also includes those forms notbiologically active. This may be accomplished by modification of themature VWF or other naturally occurring forms found in blood. The sourcefor VWF useful for the invention is mammalian, including porcine andhuman versions.

As used herein, “recombinant VWF (rVWF)” includes VWF obtained viarecombinant DNA technology. One form of useful rVWF has at least theproperty of in vivo-stabilizing, e.g. binding, of at least one FVIIImolecule and having optionally a glycosylation pattern which ispharmacologically acceptable. Specific examples thereof include VWFwithout A2 domain thus resistant to proteolysis (Lankhof et al., Thromb.Haemost. 77: 1008-1013, 1997), the VWF fragment from Val 449 to Asn 730including the glycoprotein lb-binding domain and binding sites forcollagen and heparin (Pietu et al., Biochem. Biophys. Res. Communi. 164:1339-1347, 1989). The determination of stabilizing at least one FVIIImolecule can be carried out in VWF-deficient mammals according tomethods known in the state in the art. For example, as described inExample 8 below, VWF-deficient mice are treated intravenously via thetail vein with VWF, and the level of FVIII activity in their plasma isfollowed over time. The level of FVIII activity can be measured by, forinstance, a chromogenic assay such as published in the EuropeanPharmacopoeia (Ph. Eur., 3^(rd) Ed. 1997:2.7.4).

The sample, containing FVIII (FVIII:C) is mixed with thrombin, activatedfactor IX (FIXa), phospholipids and factor X (FX) in a buffer containingcalcium. FVIII is activated by thrombin and subsequently forms a complexwith phospholipids, FIXa and calcium ions. This complex activates FX toFXa, which in turn cleaves a chromogenic substrate (e.g.(AcOH*CH₃OCO-D-CHA-Gly-Arg-pNA). The time course of para-nitroaniline(pNA) released is measured at 405 nm. The slope of the reaction isproportional to the FVIII concentration in the sample.

The rVWF of the present invention may be produced by any method known inthe art. One specific example is disclosed in WO86/06096 published onOct. 23, 1986 and U.S. patent application Ser. No. 07/559,509, filed onJul. 23, 1990, in the name of Ginsburg et al., which is incorporatedherein by reference with respect to the methods of producing recombinantVWF. This may include any method known in the art for (i) the productionof recombinant DNA by genetic engineering, e.g. via reversetranscription of RNA and/or amplification of DNA, (ii) introducingrecombinant DNA into procaryotic or eucaryotic cells by transfection,e.g. via electroporation or microinjection, (iii) cultivating saidtransformed cells, e.g. in a continuous or batchwise manner, (iv)expressing VWF, e.g. constitutively or upon induction, and (v) isolatingsaid VWF, e.g. from the culture medium or by harvesting the transformedcells, in order to (vi) obtain purified rVWF, e.g. via anion exchangechromatography or affinity chromatography.

The rVWF can be produced by expression in a suitable prokaryotic oreukaryotic host system characterized by producing a pharmacologicallyacceptable VWF molecule. Examples of eukaryotic cells are mammaliancells, such as CHO, COS, HEK 293, BHK, SK-Hep, and HepG2. There is noparticular limitation to the reagents or conditions used for producingor isolating VWF according to the present invention and any system knownin the art or commercially available can be employed. In a preferredembodiment of the present invention rVWF is obtained by methods asdescribed in the state of the art.

A wide variety of vectors can be used for the preparation of the rVWFand can be selected from eukaryotic and prokaryotic expression vectors.Examples of vectors for prokaryotic expression include plasmids such aspRSET, pET, pBAD, etc., wherein the promoters used in prokaryoticexpression vectors include lac, trc, trp, recA, araBAD, etc. Examples ofvectors for eukaryotic expression include: (i) for expression in yeast,vectors such as pAO, pPIC, pYES, pMET, using promoters such as AOX1,GAP, GAL1, AUG1, etc; (ii) for expression in insect cells, vectors suchas pMT, pAc5, pIB, pMIB, pBAC, etc., using promoters such as PH, p10,MT, Ac5, OplE2, gp64, polh, etc., and (iii) for expression in mammaliancells, vectors such as pSVL, pCMV, pRc/RSV, pcDNA3, pBPV, etc., andvectors derived from viral systems such as vaccinia virus,adeno-associated viruses, herpes viruses, retroviruses, etc., usingpromoters such as CMV, SV40, EF-1, UbC, RSV, ADV, BPV, and β-actin.

The FVIII useful with the present invention includes those forms, whichare biologically active including the full length FVIII and anyderivative capability of acting as a cofactor in the activation ofcoagulation FIX and the capability of forming a complex with VWF. TheFVIII used according to the present invention may be a plasma-derivedFVIII (pdFVIII) or a recombinant FVIII (rFVIII) or biologically activederivatives thereof. The pdFVIII and the rFVIII may be produced by anymethod known in the art. PdFVIII may be purified by any suitable means.One useful method is described in U.S. Pat. No. 5,470,954, which isincorporated herein by reference. RFVIII proteins may be prepared by anysuitable means. Examples of such rFVIII include RECOMBINATE and ADVATEboth manufactured and sold by Baxter Healthcare Corporation; REFACTO, aB-domain deleted form of FVIII manufactured and sold by WyethCorporation; and KOGENATE, manufactured and sold by Bayer Corporation.Methods and examples of rFVIII are described in U.S. Pat. Nos.4,757,006; 4,965,199; and 5,618,788, all of which are incorporatedherein by reference.

As used herein, “physiologically acceptable polymer” includes polymerswhich are soluble in an aqueous solution or suspension and have nonegative impact, such as side effects, to mammals upon administration ofthe polymer-VWF-conjugate in a pharmaceutically effective amount. Thereis no particular limitation to the physiologically acceptable polymerused according to the present invention. The polymers are typicallycharacterized as having preferably from 2 to about 300 repeating units.Examples of such polymers include, but are not limited to, poly(alkyleneglycols) such as polyethylene glycol (PEG), poly(propylene glycol)(PPG), copolymers of ethylene glycol and propylene glycol and the like,poly(oxyethylated polyol), poly(olefinic alcohol),poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),poly(hydroxyalkylmethacrylate), poly(saccharides), poly(α-hydroxy acid),poly(vinyl alcohol), polyphosphazene, polyoxazoline,poly(N-acryloylmorpholine), and combinations of any of the foregoing.

The physiologically acceptable polymer is not limited to a particularstructure and can be linear (e.g. alkoxy PEG or bifunctional PEG),branched or multi-armed (e.g. forked PEG or PEG attached to a polyolcore), dendritic, or with degradable linkages. Moreover, the internalstructure of the polymer can be organized in any number of differentpatterns and can be selected from the group consisting of homopolymer,alternating copolymer, random copolymer, block copolymer, alternatingtripolymer, random tripolymer, and block tripolymer.

These polymers also include poly(alkylene oxide) polymers, poly(maleicacid), poly(DL-alanine), such as carboxymethylcellulose, dextran,hyaluronic acid and chitin, and poly(meth)acrylates.

In an embodiment of the present invention, the physiologicallyacceptable polymer is PEG and derivatives thereof. The PEG side chaincan be linear, branched, forked or can consist of multiple arms. Thereis no specific limitation of the PEG used according to the presentinvention. A particularly useful PEG has a molecular weight in the rangeof 3,000-20,000. There are several useful PEG molecules that are in thepublic domain, i.e. that were never patented or are now off-patent.Other useful PEG molecules are disclosed in WO 03/040211; U.S. Pat. No.6,566,506; U.S. Pat. No. 6,864,350; and U.S. Pat. No. 6,455,639, forexample.

In another embodiment of the present invention, the physiologicallyacceptable polymer is polysialic acid (PSA) and derivatives thereof. PSAcan be bound to VWF by the method described in U.S. Pat. No. 4,356,170,which is herein incorporated by reference. In one embodiment of theinvention the polysaccharide compound may be naturally occurringpolysaccharide, a derivative of a naturally occurring polysaccharide, ora naturally occurring polysaccharide derivative. The polysaccharideportion of the compound has more than 5, typically at least 10, and inanother embodiment at least 20 to 50 sialic acid residues in the polymerchain. Readily available polysaccharide compounds may have up to 500saccharide residues in total, but usually have fewer than 300 residuesin the polymer chain. Generally, all of the saccharide residues in thecompound are sialic acid residues.

The polysialic acid portion at least of the polysaccharide compound, andin one embodiment the entire compound, is highly hydrophilic.Hydrophilicity is conferred primarily by the pendant carboxyl groups ofthe sialic acid units, as well as the hydroxyl groups. The saccharideunit may contain other functional groups, such as, amine, hydroxyl orsulphate groups, or combinations thereof. These groups may be present onnaturally occurring saccharide compounds, or introduced into derivativepolysaccharide compounds.

Polysaccharide compounds of particular use for the invention are thoseproduced by bacteria. Some of these naturally occurring polysaccharidesare known as glycolipids. It is particularly advantageous if thepolysaccharide compounds are substantially free of terminal galactoseunits, which tend to be recognized by galactose receptors of hepatocytesand Kupffer cells.

The VWF multimers may be covalently linked to the polysaccharidecompounds by any of various techniques known to those of skill in theart. Examples include linkage through the peptide bond between acarboxyl group on one of either the VWF or polysaccharide and an aminegroup of the other, or an ester linkage between a carboxyl group of oneand a hydroxyl group of the other. Alternatively a Schiff base can beformed between an amino group of one and an aldehyde group of the other.Other mechanisms of linkage are within the ordinary skill of the art.Various examples are identified at column 7, line 15, through column 8,line 5 of U.S. Pat. No. 5,846,951, all of which are incorporated byreference.

As used herein, reference to the VWF being bound to one or morephysiologically acceptable polymer molecules includes any suitablechemical binding, such as, covalently bound or non-covalently bound suchas ionic, hydrophobic, affinity, bioaffinity interactions. The polymercan also be coupled to the protein by use of bifunctional reagents andvia a spacer arm. In addition the polymer molecule can be coupled to theVWF by affinity interaction. For example, the VWF can be biotinylatedand avidin or strepavidin conjugated polymers can be bound to the VWF.In addition polyclonal or monoclonal anti-VWF antibodies as well asfragments thereof can be bound to a polymer and then this complex can bebound to the VWF. Polymers can be bound to the VWF also by enzymaticalmethods such as, for example, the transfer of saccharides withpolyglycosyltransferase as taught in U.S. Pat. No. 6,379,933 orglycopegylation as taught in US 2004 0132640 A1, all of which teachingsare incorporated herein by reference. Another approach is the binding ofpolymers to the VWF on the basis of their biological function like thebinding of PEGylated collagens or collagen fragments to the A1 and A3domains of the VWF. For this purpose collagens from type I and III, e.g.from human placenta, showing a strong interaction with the VWF can beused. The binding of the polymers may be stable or reversible after invivo application of the proteinaceous construct.

As used herein. “PE VWF” includes VWF which is bound to one or morePEGs, and as used herein “PEGylation” includes the process of bindingone or more PEGs to VWF. Suitable methods of PEGylation are disclosed inU.S. Pat. Nos. 5,122,614 and 5,539,063, all of which PEGylation methodsare incorporated by reference.

According to an embodiment of the present invention the physiologicallyacceptable polymer is PEG or a PEG derivative, which is covalentlylinked to VWF by any strategy and method known in the art. The mostcommon modification strategies are the binding of at least one polymermolecule via amino groups of lysine residues, the binding of at leastone polymer molecule via carbohydrate side chains, the binding of atleast one polymer molecule via sulfhydryl groups, the binding of atleast one polymer molecule via carboxyl groups of aspartic acids andglutamic acids as well as the binding of at least one polymer moleculeof hydroxyl groups and the binding of at least one polymer molecule ofthe N-terminus.

In one embodiment of the present invention the binding of at least onepolymer molecule to VWF can be performed by covalently coupling saidpolymer molecule to the amino groups of the lysine side chains of VWF.Human VWF contains free 108 lysine residues with NH₂ groups in the sidechains, which are susceptible to binding of at least one polymermolecule. Examples of lysine residues of VWF to which the polymermolecule may be covalently linked according to the present invention areshown in FIG. 1A. Suitable PEG derivatives which may be covalentlylinked to the lysine residues of VWF are, for example, polyethyleneglycols with an active N-hydroxysuccinimide ester (NHS) such assuccinimidyl succinate, succinimidyl glutarate or succinimidylpropionate, which react with the lysine residues under mild conditionsby forming an amide bond. Other examples of activated PEG are those withan active carbonate such as succinimidyl carbonate (SC-PEG) andbenzotriazole carbonate (BTC-PEG) (see page 463 of Roberts et al.,Advanced Drug Delivery Reviews 54: 459-476, 2002). SC-PEG and BTC-PEGreact preferentially with lysine residues to form a carbamate linkage,but are also known to react with histidine and tyrosine residues.Alternative methods are the binding of at least one polymer moleculewith PEG carbonates forming urethane bonds or with aldehydes or ketonesforming secondary amines, e.g. after reduction with sodium cyanoborohydride. Other PEG acylating reagents which produce urethane linkedproteins include p-nitrophenyl carbonate, trichlorophenyl carbonate, andcarbonylimidazole. These reagents are prepared by reactingchloroformates or carbonylimidazole with the terminal hydroxyl group onmonomethoxyPEG (mPEG) (see p. 464 of Roberts et al., above). Anotherexample relates to so-called “second generation” PEGylation chemistrywhereby mPEG-propionaldehyde, under acidic conditions, is selective forthe N-terminal a-amine (see page 464 of Roberts et al., above).Releasable PEG reagents such as PEG maleic anhydride, mPEG phenyl ethersuccinimidyl carbonates, and mPEG benzamide succinimidylcarbonates, maybe used to produce a conjugate which, under physiological conditions,will release the therapeutic protein with “no tag”, as described on page469 of Roberts at al., above.

In a further embodiment of the present invention, VWF can be also boundto at least one polymer molecule via its carbohydrate residues. This canbe carried out by e.g. mild oxidation of the carbohydrate chains, suchas with NaIO₄, forming an aldehyde function and subsequent coupling to aPEG, such as PEG-hydrazide. An advantage of this procedure is based onthe fact that the A1 and A3 loops of VWF, which comprise the collagenbinding sites and therefore are critical regions for the biologicalactivity of VWF, contain no carbohydrate residues and might not bemodified by this procedure as can be seen in FIG. 1B. This figure showsan example of the asparagine N-linked linked GlcNAc and the threonine-or serine O-linked GalNAc residues, which can be oxidized andsubsequently bound by at least one polymer molecule. Due to the factthat the carbohydrates of the VWF are clustered in certain domains, theVWF might be site directed bound by at least one polymer molecule by useof this modification procedure. The binding of polymer to thecarbohydrate residue is particularly advantageous when thetherapeutically active form of the VWF is desired. This can be enhancedby selective reaction of the polymer to either the N-linked or O-linkedresidues through known methods. For instance, the enzyme glucose oxidasecan be used to oxidate carbohydrate residues on VWF to generate multiplereactive aldehyde groups, which can be reacted with PEG-hydrazide toproduce a hydrazone linkage or with PEG-amine to produce a reversibleSchiff base (see page 467 of Roberts et al., above). Under certainconditions, an N-terminal serine or threonine of VWF can be used forsite-specific conjugation by converting it to a glyoxylyl derivative byperiodate oxidation (see page 467 of Roberts et al., above).

Another embodiment of the present invention is the binding of at leastone polymer molecule to VWF via sulfhydryl groups. Human VWF has 177free SH-groups, which can be modified, for example, by PEG maleimideforming a stable sulfide. PEGylation of cysteine residues may also becarried out using, for instance, PEG-vinylsulfone, PEG-iodoacetamide, orPEG-orthopyridyl disulfide (see page 466 of Roberts et al., above).

According to its multiple functions the VWF molecule has several bindingsites to specific receptors or ligands (Girma et al., Thromb Haemost.74:156-60, 1995). One important binding site is the FVIII bindingdomain, which is located at the N-terminus of the mature subunit (aminoacids 1-272). This epitope can be protected by incubation with freeFVIII and formation of a FVIII/VWF complex. Subsequently the complex ischemically modified (e.g. PEGylated or polysialylated) and thepolymer-conjugated VWF with free FVIII binding site is separated fromthe FVIII (e.g. by size exclusion chromatography with 0.3 M CaCl₂ or 2MNaCl). Similarly the VWF can be bound to an affinity resin withimmobilized FVIII. Subsequently the VWF is chemically conjugated with apolymer (e.g. a polyethylene glycol or polysialic acid derivative) andeluted from this matrix (e.g. under high salt conditions such as 0.3 MCaCl2 or 2 M NaCl) in a batch mode or by use of a chromatography column.

The FVIII binding epitope of VWF is nearly identical with theheparin-binding site. Thus, the FVIII binding site can be blocked andprotected by binding of VWF to heparin or to an affinity resin withimmobilized heparin during the chemical modification procedure.

As used herein, the term “Factor VIII-binding-site-protecting agent”refers to any agent which binds to the FVIII binding domain or epitopeon the VWF molecule. A Factor VIII-binding-site-protecting agent may beselected from Factor VIII, derivatives of FVIII, heparin, andderivatives of heparin.

The present invention is directed to increasing in vivo half-life of VWFor biologically active derivatives thereof as compared to the in vivohalf-life of VWF not linked to at least one physiologically acceptablepolymer molecule. In one embodiment of the present invention, the invivo half-life of VWF is prolonged for at least a factor of two, whilein another embodiment the in vivo half-life is increased for at least afactor of three. In still another embodiment the in vivo half-life isincreased by a factor of five by binding of at least one physiologicallyacceptable polymer molecule. The increase or prolonging of VWF half-lifecan be assessed by measuring the pharmacokinetics of VWF in FVIIIdeficient mice, as described in Example 7 below. Briefly. FVIIIdeficient mice are treated with a bolus injection of VWF premixed withFVIII via the tail vein, and VWF antigen levels are measured in plasmasamples at various time points. VWF antigen, as well as FVIII antigen,can be measured via ELISA assay.

A further aspect of the present invention relates to a complex formedbetween at least one polymer-VWF-conjugate and at least one FVIIImolecule, wherein the in vivo half-life of the FVIII is prolonged in theblood of a mammal by the polymer-VWF-conjugate.

The binding of the polymer-VWF-conjugate with FVIII prolongs orincreases the in vivo half-life of said FVIII as compared to the in vivohalf-life of a FVIII forming a complex with VWF not linked to at leastone physiologically acceptable polymer molecule. In an embodiment of thepresent invention, the in vivo half-life of FVIII is prolonged for atleast a factor of 1.5, in another embodiment for at least a factor oftwo, in another embodiment for at least a factor of three, and in afurther embodiment for at least a factor of five.

The polymer-VWF-conjugates of the present invention can be used for thetreatment of hemophilia A and/or VWD or subtypes of these two diseases.In the case of VWD, the use of polymer-VWF-conjugates will be similar tocurrent substitution therapy with VWF containing concentrates by eitherregular or irregular treatment, also referred to as prophylaxisprotocols or on demand treatment respectively. Polymer-VWF-conjugatescan also be used as an adjuvant treatment of hemophilia A prophylaxis.Under these treatment circumstances polymer-VWF-conjugates will beadministered in time intervals and independently FVIII concentrates,either plasma-derived or recombinant, the same which are currently usedfor regular treatment of hemophilia A, will be given as usual, however,with prolonged treatment intervals due to the half-life prolongingcapabilities of polymer-VWF-conjugates.

In one embodiment of the invention for both the prophylaxis inhemophilia A and VWD and for treatment of acute bleeds in hemophilia Aand VWD, a polymer-VWF-conjugate will be administered together withFVIII or in the form of a complex with FVIII to patients with hemophiliaA or VWD. In such cases arrest of internal or external bleedings needsto be achieved immediately by raising the otherwise low plasma levels ofeither VWF and/or FVIII to therapeutically effective levels. Thepolymer-VWF-conjugates can also be used for immunotolerance therapy toeradicate inhibitory antibodies, which have developed against FVIII, aclinical situation also known as inhibitor hemophilia, or against VWF.Under such circumstances supra-physiological and supra-pharmacologicallevels of FVIII are administered to patients having developed inhibitorsagainst either FVIII or VWF. This form of therapy is facilitated byapplication of polymer-VWF-conjugates having usually higher recoveriesand longer persistence in the circulation of patients receiving saidpreparations than non-conjugated VWF.

According to the state of the art in therapy and according tointernational guidelines and regulations, the pharmacokinetics ofinfused FVIII are recognized and accepted as valid surrogate markers forefficacy. This is based on the validated assumption that an infusedFVIII product which had been characterized by standardized tests forfunctional activity will be found in the blood stream and will act thereas expected as a cofactor of the tenase-complex, the activation complexof factor X by binding to FIXa and phospholipids. (Elödi et al., Thromb.Res. 21: 695-700, 1981). Therefore any pharmacokinetic analysis inanimal models will be predictive for efficacy expected in patientstreated with FVIII products.

For the determination of FIX cofactor activity a FVIII or FVIIIa sample(FVIII completely activated with thrombin) is added to a preparedmixture of FIXa, FX, phospholipid and CaCl₂. This reaction mix isincubated at 37° C. to allow complex formation and subsequent FXageneration. Subsamples are drawn at intervals up to 20 minutes and addedto a chromogenic substrate, which is selectively split by FXa. After 15minutes of incubation, the reaction is terminated by the addition ofacetic acid. The absorbance (A₄₀₅) values, which are proportional to theFXa concentrations, are measured in an ELISA reader and plotted againstthe incubation time in the reaction mix.

A further aspect of the present invention is the provision of a methodfor prolonging the in vivo half-life of FVIII in the blood of a mammalhaving a bleeding disorder associated with functional defects of ordeficiencies of at least one of FVIII and VWF, comprising the steps of:

-   a) providing at least one proteinaceous construct as defined above;-   b) providing at least one FVIII as defined above; and-   c) forming a complex between said proteinaceous construct and said    FVIII.

In one embodiment of the above method the complex of step (c) is formed“extracorporeal” (i.e. outside of the body of a mammal) by e.g. mixingthe proteinaceous construct and said FVIII, and then administering thethus formed complex in an effective amount to the mammal having saidbleeding disorder.

In a further embodiment of the above method, the complex of step (c) isformed intracorporeal (i.e. inside of the body of a mammal) between theproteinaceous construct and endogenous FVIII present in the blood of amammal having said bleeding disorder upon administering theproteinaceous construct in an effective amount to said mammal.

In yet another embodiment of the above method the complex of step (c) isformed “intracorporeal” between the proteinaceous construct andexogenous FVIII present in the blood of a mammal having said bleedingdisorder upon administering the proteinaceous construct in an effectiveamount to said mammal. Exogenous FVIII may be administeredsimultaneously in an effective amount with said proteinaceous constructor sequentially, i.e. before or after administering said proteinaceousconstruct.

As used herein, “endogenous FVIII” includes FVIII which originates fromsaid mammal. It also includes FVIII transcribed from a transgene or anyother foreign DNA present in said mammal. As used herein, “exogenousFVIII” includes FVIII which does not originate from said mammalincluding pdFVIII and rFVIII as outlined above, as well as pdFVIII whichis re-administered to the mammal from which it was isolated afterforming the above-defined complex, and rFVIII which is administered tothe mammal whose DNA has been used in the production of said rFVIII.

As used herein, “effective amount” includes a dose suitable for treatinga mammal having a bleeding disorder as outlined above; for example, forhumans preferably in a range from 5 to 1,000 IU per infusion and morepreferably in a range from 10 to 250 IU per infusion.

The route of administration does not exhibit particular limitations, andin one embodiment the proteinaceous construct or the complex of thepresent invention may be administered by injection, such as intravenous,intramuscular, or intraperitoneal injection.

The present invention is also directed at treating bleeding disordersassociated with functional defects of or deficiencies of at least one ofFVIII and VWF as used herein, including bleeding disorders wherein thecause of the bleeding disorder may be selected from the group consistingof a shortened in vivo half-life of FVIII and/or VWF, altered bindingproperties of FVIII and/or VWF, genetic defects of FVIII and/or VWF, anda reduced expression of FVIII and/or VWF. In one embodiment of thepresent invention, the bleeding disorder is selected from the groupconsisting of hemophilia A. VWD, or other diseases associated with animpaired function of VWF or impaired interaction of VWF with othermolecules.

Further, the present invention relates to a pharmaceutical compositioncomprising an effective amount of a proteinaceous construct as definedabove or an effective amount of a complex as defined above. Thepharmaceutical composition may further comprise a pharmaceuticallyacceptable carrier, diluent, salt, buffer, or excipient. Thepharmaceutical composition can be used for treating the above-definedbleeding disorders. The pharmaceutical composition of the invention maybe a solution or a lyophilized product. There are many known methods offorming stable solutions of proteins, and specifically VWF and FVIII.For example, U.S. Pat. Nos. 6,586,573; 5,565,427; 5,763,401; 5,733,873;4,877,608; 5,605,884; and 5,328,694. These solutions can be subjected toany suitable lyophylization process, for example, the process describedin U.S. Pat. No. 6,586,573, which is herein incorporated by reference.The present invention includes other suitable forms of thepolymer-VWF-conjugate either alone or in combination with FVIII.

The figures show:

FIGS. 1A and 1B show the schematic structure of VWF/with examples fortarget sites for conjugation. The grey dots in FIG. 1A indicate thelysine residues of VWF which can be bound by at least one polymermolecule, and FIG. 1B shows the carbohydrate residues in VWF which canbe bound by at least one polymer molecule.

FIG. 2 shows the pharmacokinetics of a polymer-VWF-conjugate compared tonon-conjugated VWF in mice with VWD.

FIG. 3 shows the pharmacokinetics of a polymer-VWF-conjugate compared tonon-conjugated VWF in hemophilic mice.

FIG. 4 shows the pharmacokinetics of FVIII in VWD mice treated with rVWFor PEG-rVWF.

FIG. 5 shows the recovery of rFVIII in hemophilic mice after applicationof rFVIII complexed with polymer-VWF-conjugate and rFVIII complexed withVWF.

FIG. 6 shows the recovery of VWF in hemophilic mice after application ofrFVIII complexed with polymer-VWF-conjugate and rFVIII complexed withVWF.

FIG. 7 shows the pharmacokinetics of rFVIII and PEG-rVWF in FVIII×VWFdouble knockout mice.

FIG. 8 shows the pharmacokinetics of rFVIII and PEG-rVWF incrossbreed-mice.

FIG. 9 shows VWF:Ag increase in mouse plasma (lysine and carbohydratecoupling).

FIG. 10 shows a biomolecular interaction study (left diagram) andcomparison of binding capacity of FVIII (right diagram) ofPEG-conjugated rVWF compared to non-conjugated rVWF.

FIG. 11 shows the mass increase of VWF after polymer conjugationmeasured by SDS-PAGE.

FIG. 12 shows the mass increase of VWF after polymer conjugationmeasured by agarose electrophoresis to analyze VWF multimers.

FIG. 13 shows the pharmacokinetics of rFVIII in FVIII-K.O.-mice afterapplication of rFVIII complexed with polymer-VWF-conjugate (rVWF withPEGylated lysine residues) and rFVIII complexed with rVWF.

FIG. 14 shows the pharmacokinetics of rVWF in FVIII-K.O.-mice afterapplication of rFVIII complexed with polymer-VWF-conjugate (rVWF withPEGylated lysine residues) and rFVIII complexed with rVWF.

FIG. 15 shows the determination of the FVIII binding capacity ofPEG-conjugated rVWF compared to non-conjugated rVWF using a combinedELISA and chromogenic assay (ECA).

FIG. 16 shows the determination of the FVIII binding capacity ofPEG-conjugated rVWF using the surface plasmon resonance technology.

FIG. 17 shows the mass increase of VWF after polymer conjugationmeasured by SDS-PAGE as described in Example 13.

FIG. 18 shows the determination of the FVIII binding capacity offurin-maturated rVWF compared to a pdVWF and non-treated rVWF using acombined ELISA and chromogenic assay (ECA).

FIG. 19 shows the determination of the FVIII binding capacity offurin-maturated rVWF compared to a pdVWF and non-treated rVWF using thesurface plasmon resonance technology.

FIG. 20 shows the isoelectric focusing patterns of rVWF-polysialic acidconjugate and rVWF under reducing conditions.

FIG. 21 shows the pharmacokinetics of rVWF-polysialic acid conjugate andrVWF in VWF-deficient mice.

FIG. 22 shows the time course of mouse FVIII activity after applicationof rVWF-polysialic acid conjugate or rVWF in VWF-deficient mice.

FIG. 23 shows the pharmacokinetics of PEG-rVWF (branched PEG 20K SG) andrVWF in FVIII deficient mice.

FIG. 24 shows the pharmacokinetics of rFVIII, co-infused with PEG-rVWF(branched PEG 20K SG) or rVWF in FVIII deficient mice.

FIG. 25 shows the pharmacokinetics of rFVIII, co-infused with variousamounts of PEG-rVWF (branched PEG 20K SG) in FVIII deficient mice.

FIG. 26 shows the pharmacokinetics of PEG-rVWF #A (5 mg PEG/mg protein),PEG-rVWF #B (20 mg PEG/mg protein) and native rVWF in FVIII deficientmice.

FIG. 27 shows the pharmacokinetics of rFVIII, co-infused with PEG-rVWF#A (5 mg PEG/mg protein), PEG-rVWF #B (20 mg PEG/mg protein) or nativerVWF.

The present invention will be further illustrated in the followingexamples, without any limitation thereto.

EXAMPLES Example 1 Preparation of Polymer—VWF—Conjugate by Modificationof Carbohydrate Residues

For preparation of polymer—VWF conjugate via carbohydrate residues (FIG.1B) a solution of rVWF (final concentration: 500 μg/ml) was prepared in20 mM sodium acetate buffer, pH 6.0 and NaIO₄ was added (finalconcentration 5 mM) for the oxidation of carbohydrate residues. Theoxidation was carried out for 20 min at 4° C., then sodium bisulfite(final concentration 5 mM) was added to stop the reaction. SubsequentlymPEG-hydrazide (chain length: 3 kD) was added (final concentration 10mM) and the PEGylation of the VWF was performed for 1 h at roomtemperature. Then the PEGylated VWF was purified by size-exclusionchromatography. The reaction mixture was applied onto a chromatographiccolumn (size: 26 mm×840 mm) filled with Sephacryl S-300 HR (Amersham)and the PEGylated VWF was separated from the reagents by using 20 mMHEPES-buffer, 150 mM NaCl, pH 7.4 containing 5% trehalose. The modifiedVWF was eluted in the void volume as indicated by measurements ofVWF-antigen levels and OD 280 nm. The VWF containing fractions weredirectly applied to an anion-exchange column (size: 10 mm×108 mm) filledwith EMD TMAE 650 M (Merck) for further purification. Than the PEGylatedVWF was eluted with 20 mM HEPES buffer, containing 5% trehalose and 1000mM NaCl.

Example 2 PEGylation of Lysine Residues in VWF with mPEG SuccinimidylSuccinate

For PEGylation of VWF via lysine residues (FIG. 1A) a solution of rVWF(final concentration: 500 μg/ml) was prepared in 20 mM HEPES-buffer, 150mM NaCl, pH 7.4, containing 5% sucrose) and mPEG succinimidyl succinate(chain length: 5 kD) was added (final concentration 10 mM). The VWF wasPEGylated for 1 h at room temperature. Subsequently the PEGylated VWFwas purified by size-exclusion chromatography. The reaction mixture wasapplied onto a chromatographic column filled with Sephacryl S-300 HR(Amersham) and the PEGylated VWF was separated by the same buffer systemused for the PEGylation reaction. The VWF was eluted in the void volumeas indicated by measurements of VWF-antigen levels and OD 280 nm. TheVWF containing fractions were directly applied to an anion-exchangecolumn (size: 26 mm×840 mm) filled with EMD TMAE 650 M (Merck) forfurther purification. Then the PEGylated VWF was eluted with 20 mM HEPESbuffer, containing 5% sucrose and 1000 mM NaCl.

Example 3 PEGylation of Lysine Residues in VWF with mPEG p-NitrophenylCarbonate

For PEGylation of VWF with mPEG p-nitrophenyl carbonate a solution ofplasma derived VWF (final concentration: 500 μg/ml) was prepared in 20mM HEPES-buffer, 150 mM NaCl, pH 7.6 containing 5% sucrose) and mPEGp-nitrophenyl carbonate (chain length: 2 kD) was added (finalconcentration 10 mM). The VWF was PEGylated for 2 h at room temperature.Subsequently the PEGylated VWF was purified by size-exclusionchromatography. The reaction mixture was applied onto a chromatographiccolumn filled with Sephacryl S-300 HR (Amersham) and the PEGylated VWFwas separated by the same buffer system used for the PEGylationreaction. The VWF was eluted in the void volume as indicated bymeasurements of VWF-antigen levels and OD 280 nm.

Example 4 PEGylation of Sulfhydryl Residues in VWF with mPEG Maleimide

For PEGylation of VWF via free SH residues with mPEG maleimide asolution of rVWF (final concentration: 500 μg/ml) was prepared in 20 mMHEPES-buffer, 150 mM NaCl, pH 7.6 containing 4% mannose and 1%trehalose) and mPEG maleimide (chain length: 10 kD) was added (finalconcentration 10 mM). The VWF was PEGylated for 2 h at room temperature.Subsequently the PEGylated VWF was purified by size-exclusionchromatography. The reaction mixture was applied onto a chromatographiccolumn filled with Sephacryl S-300 HR (Amersham) and the PEGylated VWFwas separated by the same buffer system used for the PEGylationreaction. The modified VWF was eluted in the void volume as indicated bymeasurements of VWF-antigen levels and OD 280 nm.

Example 5 Coupling of Dextran to VWF

A dextran (MW 40 kD) solution of 6 mg/ml was prepared in 20 mM sodiumacetate buffer, pH 6.0 and NaIO₄ was added (final concentration 10 mM)to generate free aldehyde groups. The oxidation was carried out for 1 hat 4° C. in the dark, then sodium bisulfite (final concentration 5 mM)was added to stop the reaction. The activated dextran was dialyzedagainst 0.1 M sodium phosphate buffer, pH 7.2, containing 0.15 M NaCl(PBS-buffer). Then 2.4 ml of this solution of activated dextran wereadded to 10 ml of a solution of rVWF (concentration: 0.6 mg/ml in PBSbuffer). To this mixture 5 ml of a sodium cyanoborohydride solution (64mg/ml in PBS buffer) were added and incubated at room temperatureovernight in the dark. Then 3 ml of a 1.0 M TRIS-HCl solution, pH 7.2,were added to block remaining aldehyde groups and incubated for 1 h atroom temperature and dialyzed against 20 mM HEPES-buffer, pH 7.4,containing 5% sucrose. Then the dextran coupled rVWF derivative wasfurther purified by size-exclusion chromatography by applying themixture onto a chromatography column (size: 50 mm×860 mm) filled withSephacryl S-300 HR (buffer: 20 mM HEPES, 5% sucrose, pH 7.4). The rVWFderivative was eluted in the void volume as indicated by measurements ofVWF-antigen levels and OD 280 nm. These fractions were collected andconcentrated by ultrafiltration using a 100 kD regenerated cellulosemembrane (Millipore).

Example 6 Pharmacokinetics in VWD-Mice

VWF-deficient mice described in detail by Denis et al. (PNAS 95:9524-9529, 1998) were used as a model of human VWD resembling severetype III VWD. Groups of 5 mice received a bolus injection via the tailvein either with PEG-rVWF (chain length 3 kD, PEGylation of rVWFaccording to Example 1) or native rVWF as control in a dose of 40 UVWF:Ag/kg bodyweight, based on detectable VWF (ELISA) after PEGylation.The PEG-rVWF groups were anesthetized 5 min, 30 min, 1 h, 2 h, 6 h, 10 hand 24 h after injection (5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 10h and 24 h for the control groups) and citrate plasma was prepared fromheart puncture. VWF antigen levels were followed in plasma. The resultsof this experiment are summarized in FIG. 2. Native rVWF is eliminatedfrom the circulation in a typical biphasic manner, as described in theliterature (Lenting et al., J. Biol. Chem. 279: 12102-12109, 2004) andfalls below the limit of detection between 600 min and 1440 min,equivalent to 10 and 24 h. In contrast, PEGylated rVWF after an initialincrease from 0 at the time of injection to approximately 0.6 U per mlplasma 10 h after injection was still present to a substantially higherlevel of approximately 0.4 U per ml even 24 h after injection with aflat decline between 10 h and 24 h indicating a much longer persistenceof PEGylated rVWF. The gradual increase in measurable VWF over timeindicates the reversibility of the conjugation of the polymer PEGconjugated with VWF, which after release from the polymer becomesaccessible for measurement. The long circulation time of PEG-VWF in thismodel demonstrates that this preparation can be used for theprophylactic treatment of VWD.

Example 7 Pharmacokinetics in FVIII-K.O.-Mice

FVIII deficient mice described in detail by Bi et al. (Nat. Genet. 10:119-121, 1995) were used as a model of severe human hemophilia A. Groupsof 5 mice received a bolus injection (13 ml/kg) via the tail vein witheither PEG-rVWF (chain length 3 kD, PEGylation of rVWF was performedaccording to example 1), or native rVWF, each premixed with rFVIII toachieve 3 U FVIII/ml and 3 U VWF:Ag/ml. After anesthesia, citrate plasmawas prepared by heart puncture from the respective groups, 5 min and 6 hafter product injection. A control group received buffer and was bled 5min after injection. VWF antigen levels were measured in plasma samples.The results of this experiment are summarized in FIG. 3. The curvesindicate a typical elimination for rVWF dropping to near base-level ofVWF, present in FVIII deficient mice, whereas after application ofPEGylated rVWF, levels increased within the observation period of 6 h.This again indicates the reversibility of the conjugation of the polymerPEG conjugated with VWF, which after release from the polymer becomesaccessible for measurement and persists to increase even after 360 min(6 h) following application.

Example 8 FVIII Increase in VWD-Mice

Von Willebrand deficient mice described in detail by Denis et al. (PNAS95: 9524-9529, 1998) were used as a model of human VWD resembling severetype III VWD. Groups of 4-5 mice were treated intravenously via the tailvein with PEG-rVWF (chain length 5 kD, PEGylation of rVWF was performedaccording to Example 2) in 20 mM HEPES, 150 mM NaCl, 5% saccharose pH7.4 or with native rVWF. A control-group was treated with buffer.(PEG-rVWF-dose 18 U VWF:Ag/kg based on ELISA, 2700 μg/kg, rVWF nativedose: 2400 μg/kg).

Each mouse received a volume dose of 10 ml/kg. At time points afterinjection of PEG-rVWF (5 min, 1 h, 3 h, 6 h, 10 h, 24 h and 48 h) ornative rVWF (5 min, 15 min, 30 min 1 h, 2 h, 6 h, 24 h and 32 h), groupsof 4-5 mice were anesthetized, citrate plasma was prepared from heartpuncture and the level of FVIII activity (chromogenic in house assay)was followed in plasma. The control-group was bled 15 minutes afterinjection. The results of this experiment are summarized in FIG. 4.

The level of endogenous FVIII in mice increases as a result of rVWFinfusion. The area under curve (AUC) after application of PEGylated rVWFwas 8.0 U*h/ml compared to only 3.3 U*h/ml after application of rVWF.This indicates a substantially longer circulation time for PEGylatedrVWF. The results show that the PEGylated VWF can be used for theprophylactic treatment of secondary FVIII deficiency in VWD.

Example 9 Recovery of rFVIII and VWF in FVIII-K.O.-Mice

FVIII deficient mice described in detail by Bi et al. (Nat. Genet. 10:119-121, 1995) were used as a model of severe human hemophilia A. Groupsof 5 mice received a bolus injection (13 ml/kg) via the tail vein witheither PEG-rVWF (HZ-PEG, 3K, coupled via carbohydrates), or native rVWF,each premixed with rFVIII to achieve 3 U FVIII/ml. Citrate plasma byheart puncture after anesthesia was prepared from the respective groups,5 min and 6 h after injection. FVIII activity and VWF antigen recoverylevels were measured in plasma samples. The results of this experimentare summarized in FIGS. 5 and 6.

PEGylated rVWF for both preparations induced a higher recovery ofco-injected rFVIII, compared to untreated rVWF. The VWF levels increasedfor PEGylated rVWF over time, while normal rVWF was eliminated almostcompletely within 360 min. The results show that the PEGylated VWFcomplexed with FVIII can be used for acute treatment of hemophilia Awith the benefit of increased FVIII circulation time.

Example 10 Increase of FVIII Half-Life in FVIII×VWF-Double Knockout Mice

FVIII×VWF double knockout mice were obtained by cross-breeding of FVIIIdeficient and VWF-deficient mice. Those mice suffer from FVIIIdeficiency as well as from VWF deficiency, thus providing an ideal modelfor studying FVIII-VWF interactions in an animal model.

Groups of 5 FVIII×VWF double knockout mice (FVIII deficient mice werecrossbred with VWF-deficient mice) received a bolus injection (11 ml/kg)via the tail vein with either PEG-rVWF (chain length 5 kD, PEGylation ofrVWF was performed according to example 2 by modification of lysineresidues with mPEG succinimidyl succinate) in 20 mM HEPES, 150 mM NaCl,5% saccharose pH 7.4 or with native rVWF SS-PEG, or native rVWF, eachpremixed with rFVIII to achieve 9 U and 9 U VWF antigen/ml and 0.67 UVWF:RCo/ml. The VWF-antigen values were measured by use of an ELISAmethod as published (Ingerslev, Scand. J. Clin. Invest. 47: 143-149,1987). The functional VWF:RCo activity reflecting the platelet bindingproperties of the VWF in the process of primary hemostasis was measuredaccording to Macfarlane et al. (Thromb. Diath. Haemorrh. 34: 306-308,1975). Five min, 3 h, 6 h, 10 h and 24 h after injection, citrate plasmaby heart puncture after anesthesia was prepared from the respectivegroups. FVIII activity and VWF antigen levels were measured in plasmasamples.

Half-life of FVIII and VWF was calculated using the MicroMath Scientistprogram (Micromath Research, Saint Luis, Mo., US) employing onecompartment model from the pharmacokinetic library. Half-life for FVIII,co-infused with either rVWF or PEGylated rVWF increased from 1.88 h to2.58 h, the area under the curve (AUC) increased from 4.3 to 7.3 U*h/ml.The half-life of VWF increased from 3.1 to 10.4, the area under curve isincreased from 5.7 to 22.8. The results are summarized in FIGS. 7 and 8.The data show that PEG-VWF can be used for the acute and prophylactictreatment of hemophilia A and VWD with the benefit of long circulationtimes of VWD and FVIII.

Example 11 Demonstration of Reversibility of PEGylation in Mouse Plasma

The reversibility of PEGylation was demonstrated by in vitro experimentswith VWF deficient plasma. Citrated plasma was obtained fromVWF-deficient mice (Denis et al. PNAS 95: 9524-9529, 1998) bycentrifugation at 1100×g for 15 min at 4° C. Four volumes of mouseplasma were mixed with 1 volume of PEGylated rVWF prepared according toExample 1 (PEG coupling via carbohydrates) or Example 2 (PEG couplingvia lysine residues) and kept at 37° C. for 48 h. Non-PEGylated rVWF wasused as a control in both experiments. Subsamples were withdrawnimmediately after mixing and 1 h, 5.5 h, 24 and 48 h later and the VWFantigen content was assayed from frozen samples by use of a sandwichELISA system. A polyclonal anti-VWF antibody (DAKO) was used for coating96 well ELISA plates and a goat-anti-rabbit-IgG-HRP-conjugate (AXELL)was assayed for the detection of bound factor VWF. Over time, increasingamounts of VWF antigen were measured demonstrating the reversibility ofconjugation of polyethylene glycol to VWF also in ex vivo plasma samples(FIG. 9).

Example 12 Determination of FVIII Binding Capacity of PEGylated VWFPreparations

The FVIII binding capacity of different PEGylated rVWF-preparations wascompared by surface plasmon resonance experiments (Karlsson et Fait, J.Immunol. Methods 200: 121-33, 1997) using a BIACORE® 3000 instrument(BIACORE, Uppsala, Sweden). In general, ligands are immobilised to asensor chip and the binding of other components to the ligand isdetermined by surface plasmon resonance. By use of this technique thechange of the refractive index of the solution close to the surface ofthe chip is measured. A change in the concentration of a bound componentat the surface of the chip is detected as a signal, which is expressedin arbitrary resonance units (RU). There is a linear relationshipbetween the mass of protein bound to the immobilized ligand and the RUobserved. The PEGylated VWF preparations were immobilized at 25° C. tothe dextran surface of the BIACORE™ sensor chip using NHS/EDC chemistryat 7000-9000 RU and 25° C. A 10 mM HEPES buffer pH 7.4, containing 150mM NaCl, 3 mM EDTA and 0.005% surfactant P20 (HBS-buffer, BIACORE) wasused at a flow rate of 15 μl/min. The binding of different amounts of acommercially available FVIII product (ADVATE, Baxter AG, Vienna,Austria) was measured as illustrated in FIG. 10. This figuredemonstrates the FVIII binding capacity of a PEGylated rVWF preparation,modified with mPEG maleimide 5000, prepared according to Example 4. Theresults of the BIACORE experiments with different PEGylated rVWFpreparations are summarized in Table 1. In this table the differentFVIII binding capacities of the PEGylated rVWF preparations are given inpercent of the RU values of the non PEGylated reference preparation atthe maximal level of the reference in the range of 10-20 IU FVIII/ml(chromogenic assay).

TABLE 1 FVIII binding capacities of PEGylated rVWF preparations (nonPEGylated rVWF = 100%) Reagent concentration Reagent 0 mM 1 mM 5 mM 10mM 20 mM mPEG SS 5000 100% 30-40% 20-30%  0-20%     0% mPEG MAL 5000100% 70-90% 40-60% 30-50% 20-40% mPEG Hz 3000 (a) 100% 60-80% 30-50%10-20%  0-5% (a) Oxidized with 5 mNaIO₄

Example 13 Mass Increase of VWF after Polymer Conjugation

rVWF was PEGylated according to Example 2 using mPEG succinimidylsuccinate (chain length: 5 kD) in various concentrations (1 mM, 2.5 mM,5 mM, 7.5 mM and 10 mM). The PEGylated VWF species were analyzed withtwo different methods: SDS Polyacrylamide gel electrophoresis and VWFmultimer analysis. The SDS gel electrophoresis was performed underreducing conditions using a 3-8% gradient gel (Tris Acetat Gel/Bio-Rad).VWF multimer analysis was carried out according to Ruggeri at Zimmerman(Blood 57: 1140-43, 1981) using a 1.6% agarose gel. The visualization ofVWF-multimers was carried out according to Aihara et al. (Thromb.Haemost. 55: 263-67, 1986).

The SDS gel is electrophores (FIG. 11) shows a rVWF preparationconsisting of the mature VWF (lower band) and pro-VWF (upper band) andthe increase of the molecular weight after PEGylation by use ofdifferent reagent concentrations. In addition the shift of the molecularweight from a human serum albumin (HSA) preparation after PEGylation(PEGylation was carried out according to Example 2 is shown as areference preparation. It is demonstrated that the molecular weight ofHSA is shifting from 66,000 Da to 190,000 Da showing the efficacy of thePEGylation procedure.

FIG. 12 shows the multimeric pattern of rVWF before and after PEGylationwith different reagent concentrations. A broadening and a shift tohigher molecular weight of the different multimers with increasingreagent concentration is clearly demonstrated.

Example 14 PEGylation of Lysine Residues in VWF with mPEG SuccinimidylGlutarate

For PEGylation of VWF via lysine residues (FIG. 1A) a solution of rVWF(final concentration: 500 μg/ml) was prepared in 20 mM HEPES-buffer, 150mM NaCl, pH 7.4, 5% sucrose) and mPEG succinimidyl glutarate (chainlength: 5 kD) was added (final concentration: 200 mg PEG succinimidylglutarate/mg protein). Then the pH value was adjusted to 7.4 with 0.1 MNaOH. The VWF was PEGylated for 1 h at room temperature and purified asdescribed in Example 2.

Example 15 Increase of FVIII Half-Life in FVIII-K.O.-Mice

FVIII deficient mice described in detail by Bi et al. (Nat. Genet. 10:119-121, 1995) were used as a model of severe human hemophilia A. Groupsof 5 mice received a bolus injection (10 ml/kg) via the tail vein witheither PEG-rVWF (SS-PEG, 5K) prepared according to Example 2 or nativerVWF, each premixed with recombinant FVIII to achieve 10 U FVIII/ml and10 U VWF/ml. Citrate plasma by heart puncture after anesthesia wasprepared from the respective groups, 5 min, 3, 9 and 24 h afterinjection. FVIII activity and VWF antigen recovery levels were measuredin plasma samples. The results of this experiment are summarized inFIGS. 13 and 14.

Half-life for FVIII increased from 1.8 h (in the presence of nativerVWF) to 3.9 h (when applied together with PEG-rVWF), area under curve(AUC) increased from 4.1 to 7.8 U*h. VWF half-life increased from 3.2 to13.6 h, AUC for VWF was approximately quadrupled from 7.7 to 32.1 U*h.

Example 16 Determination of FVIII Binding Capacity of SS-PEGylated VWFPreparations by Different Methods

The FVIII binding capacity of different PEGylated rVWF-preparations wasmeasured by a combined ELISA and chromogenic assay system (ECA) using amodification of the method described by Bendetowicz et al. (Blood 92:529-538, 1998). Microtiter plates were coated with 200 μl of 2.6 μg/mLanti-vWF polyclonal antibody in 50 mmol/l Na₂CO₃/NaHCO₃, pH 9.6. Plateswere subsequently washed after each step with PBS-Tween buffer (100 mMNa₂HPO₄/KH₂PO₄, 150 mM NaCl, pH 7.6 and 0.05% Tween 20). Plates wereblocked for 1 h at 37° C. in 0.1% dry-milk/2 mM benzamidine inPBS-Tween. Increasing amounts of VWF were preincubated for 25 min at 37°C. with 0.2 U/ml rFVIII (ADVATE, Baxter AG, Vienna, Austria), and 100 μlof these mixtures were added to the plates. After incubation, the amountof FVIII bound to the captured VWF was measured by FVIII chromogenicassay (Technoclone, Vienna, Austria). The FVIII binding capacity hasbeen expressed as changes in the absorbance measured at 405 nm (dA405)in 1 min. FIG. 15 shows the VWF dose-dependent FVIII binding of twoPEGylated VWF preparations, both modified with mPEG-succinimidylsuccinate (SS). The FVIII binding capacity of the modified VWFpreparations was calculated as % of the unmodified starting VWFpreparations and found to be 20% for PEG-SS-rVWF-1 and 50% ofPEG-SS-rVWF-2.

FIG. 16 shows the FVIII binding capacity of the two PEG-SS-rVWFpreparations, as measured by the surface plasmon resonance method asdescribed in Example 12. The calculated binding capacity was 25 and 45%,respectively.

The appropriate increases in the molecular mass of the rVWF moleculeafter the PEG-SS conjugation measured by SDS-PAGE are demonstrated inFIG. 17.

Example 17 PEGylation of Sulfhydryl Groups in VWF with Branched PEGMaleimide

For PEGylation of VWF via free SH residues with a branched PEG maleimidea solution of recombinant VWF (final concentration: 500 μg/ml) isprepared in 20 mM HEPES-buffer, 150 mM NaCl, pH 7.6 containing 3%trehalose). Then a branched mPEG maleimide (chain length: 20 kD)supplied by NOF corporation (NOF Europe, Grobbendonk, Belgium) is added(final concentration 10 mM). The VWF is PEGylated for 2 h at roomtemperature under gentle stirring. Subsequently the PEGylated rVWF isseparated from the reagents by ultrafiltration/diafiltration (UF/DF)using a 100 kD membrane consisting of regenerated cellulose (Millipore).

Example 18 PEGylation of Carboxyl Groups in VWF with mPEG Hydrazide/EDC

A furin maturated rVWF is prepared and purified according to Example 23.The preparation is dialyzed against 50 mM phosphate buffer, pH 6.2 anddiluted to a concentration of 400 μg/ml. Then mPEG hydrazide (mPEG Hz)with a chain length of 5 kD is added (concentration: 60 mg mPEG Hz/mgVWF). 30 μl of a freshly prepared solution of 500 mM1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) is added to 1 ml ofthe VWF containing mixture and incubated at room temperature undergentle shaking for 5 h. The reagents are separated from the PEGylatedrVWF by UF/DF against 20 mM HEPES-buffer (150 mM NaCl, pH 7.4) using a100 kD membrane (regenerated cellulose/Millipore).

Example 19 Modification of Lysine Residues in VWF with Polysialic Acid

The modification of lysine residues with polysialic acid (colominicacid, CA) was carried out as described by Fernandes et Gregoriadis(Biochim. Biophys. Acta 1341: 26-34, 1997) and Jennings et Lugowski (J.Immunol. 127: 1011-1018, 1981). A solution of colominic acid(concentration: 20 mg/ml) containing 0.1 M NaIO₄ was stirred for 15 minin the dark at room temperature to oxidize the CA. Two ml ethyleneglycol per ml of the activated CA solution were added and stirred forfurther 30 min in the dark at room temperature. The solution wasdialyzed overnight against 0.05 M sodium phosphate buffer, pH 7.2 in thedark. Subsequently an aliquot of this solution was added to a rVWFsolution (400 μg/ml) in 0.05 M sodium phosphate to give a finalconcentration of 50 mg activated CA per mg VWF. This mixture was stirredfor 30 minutes at room temperature in the dark. NaCNBH₃ was added (1mg/mg rVWF) and the mixture was incubated for 18 h at room temperaturein the dark under gentle shaking. An aqueous 1 M TRIS-solution, pH 7.2was added (50 μl per mg NaCNBH₃) and stirred for 1 h to terminate thereaction. The free reagents were separated from the rVWF-polysialic acidconjugate by UF/DF using a 100 kD membrane (regeneratedcellulose/Millipore).

The FVIII binding capacity of this preparation was determined asdescribed in Example 16.

The conjugation of rVWF with polysialic acid was demonstrated by theshift of the isoelectric point (PI) detected by isoelectric focusing(IEF) under reducing conditions. FIG. 20 shows a comparison of a VWFpreparation before and after conjugation with polysialic acid accordingto this example. The Ampholine PAGplate system (pH 3.5-9.5) was usedaccording to the instruction of the manufacturer (Amersham Bioscience).The introduction of acidic groups by the conjugation with polysialicacid lead to one single acidic band for the modified rVWF.

A FVIII binding capacity of 54% as described in Example 16 wasdetermined by use of the ECA test. A FVIII binding capacity of 70% wasdetermined using the surface plasmon resonance method (Example 12).

Example 20 PEGylation Under Shear Stress Conditions

A perfusion chamber was manufactured as described by Sakariassen et al.(J. Lab. Clin. Med. 102: 522-535, 1983) and used for the PEGylation ofrVWF under shear stress conditions. A solution of rVWF (500 μg/ml) in 20mM HEPES, pH 7.4 containing 3% trehalose was prepared. Then mPEGsuccinimidyl succinate (final concentration: 5 mM) is added, the pHvalue adjusted with 0.1 M NaOH to 7.4 and immediately filled in theperfusion system. Then the PEGylation is carried out at room temperatureat a shear rate of 2500 sec⁻¹ by use of a peristaltic pump under theperfusion conditions described by Sakariassen et al. (J. Lab. Clin. Med.102: 522-535, 1983).

Example 21 Preparation of pdVWF

The preparation of pdVWF was carried out according to Thorell etBlombäck (Thromb. Res. 35: 431-450, 1984) with modifications. Forpreparation of pdVWF, 1.5 kg of cryoprecipitate was dissolved at 20-30°C. in 6 l of water. After stirring for 1 h, the fibronectin precipitatewas removed by centrifugation. 8 g NaCl per liter supernatant wereadded, the solution was warmed to room temperature and SD-stock reagent(1% Tween 80+0.18% acetyltriethylcitrate final concentration) was addedfor virus inactivation.

The solution was further purified on a EMD-TMAE Fractogel 650 M column(XK50/180) preequilibrated with 0.2M NaCl, 0.02M sodium acetate, pH 6.5(washing buffer). After washing with 20 column volumes of washingbuffer, VWF was eluted with 0.5M NaCl, 0.02M Na-citrate, pH 6.9. Forprecipitation of VWF glycine (1M final concentration) and NaCl (3M finalconcentration) were added. The precipitate was dissolved in buffer andapplied onto a Sephacryl S-400 HR (Amersham) column equilibrated in 0.3MCaCl₂, 0.15M NaCl, 20 mM HEPES, pH 7.4 for final purification.

TABLE 2 Purification of pdVWF Specific Act. VWF:CB/ VWF:RCo/ VWF:RCo/[IU VWF:Ag/ VWF:Ag VWF:Ag FVIII Ag Sample mg protein] [IU/IU] [IU/IU][IU/IU] Starting Material (n = 2) 1.3 0.35 0.17 0.33 (Cryoprecipitateresuspended) Centrifugation supernatant (n = 2) 2.3 0.34 0.19 0.39Fractogel EMD-TMAE 650M (n = 2) 191 0.72 0.47 0.77 Sephacryl S-400 HR (n= 2) 192 0.91 0.56 40

Example 22 PEGylation of Lysine Residues in pdVWF with PEG SS

PdVWF was prepared according to Example 21 and diluted with 20 mMHEPES-buffer pH 7.4 (containing 150 mM NaCl and 3% sucrose) to a finalconcentration of 400 μg/ml. Then mPEG succinimidyl succinate (chainlength: 5 kD) was added (concentration: 10 mg PEG SS 5000/mg VWF) andthe pdVWF was PEGylated for 1 h at room temperature. Then the reagentwas separated from the PEGylated VWF by UF/DF using a 100 kD membraneconsisting of regenerated cellulose (Millipore).

Example 23 Furin Maturation and Purification of the Furin-Maturated rVWF

143 kg of the flow-through fraction of an anti-FVIII antibody columnderived from a rFVIII fermentation and purification process were treatedwith furin for removal of the propeptide as described by Schlokat et al.(Biotechnol. Appl. Biochem. 24: 257-267, 1996) and were sterilefiltered. The process was based on earlier work (Fischer et al., FEBSLett. 375: 259-262, 1995; Fischer et al. PCT/AT98/00034[WO 98/38219],1-33, 1998, 18 Feb. 1998 and Kaersgaard et Barington, J. Chromatogr. B715: 357-367, 1998).

After 4-fold dilution with water to 16 mS/cm 633 kg of diluted solutionwere applied on a XK50/15 EMD-TMAE-Fraktogel 650M-column (300 ml gel;Merck; #K14540281) equilibrated with 10 mM Tris, 100 mM NaAc, 86 mMNaCl, pH 6.5, mS/cm (equilibration buffer). The column was washed withequilibration buffer and eluted with 100 mM NaAc, 250 mM NaCl, 100 mMglycine, 3 mM CaCl₂

4506 g of TMAE-eluate were filtered on Sartoclean GF (0.8 & 0.65μ) andSartobran P (0.45 & 0.2μ), diluted 1.5 fold to 29 mS/cm and pumpedthrough a Mustang Q filter (#IH18770932) for removal of DNA. After SDtreatment for 60 min at 22+/−2° C. and 2-fold dilution with water to 16mS/cm, the solution was applied to an Amicon 70/29 UNOsphere S-column(600 ml gel; Bio-Rad, #78960C) equilibrated with equilibration buffer ofthe TMAE step. Column was washed with equilibration buffer and elutedwith elution buffer of the TMAE step.

3223 g of UNO-S eluate were concentrated 15 fold by ultrafiltrationusing a 30 kDa 0.1 m²-membrane (Hydrosart #01080217, Sartorius)consisting of regenerated cellulose. 201 g of the concentrate wasfinally purified by size exclusion chromatography on a XK50/86.5Superose 6 Prep Grade column equilibrated with 100 mM NaAc, 500 mM NaCl,pH 7.0 (1698 ml gel; GE Healthcare #17-0489-01).

TABLE 3 Purification of furin-processed rVWF Specific Act. VWF:CB/ CHO/Purification Purification VWF:RCo/ [IU VWF:Ag/ VWF:Ag VWF:Ag factorfactor per FVIII Ag Sample mg protein] [IU/IU] [ng/IU] (CHO) step (CHO)[IU/IU] Starting material (n = 4) 1 0.75 23678 1.0 1 0.69 1. TMAE Eluate(n = 5) 34 0.84 2766 8.6 8.6 0.62 Mustang Q (n = 4) 44 0.70 2405 10 1.10.47 UNO-S Eluate (n = 4) 121 0.71 329 72 7.3 0.40 Superose 6 PG (n = 2)160 0.99 4 6150 85 0.39

Example 24 PEGylation of Furin-Maturated rVWF

Furin-maturated VWF was prepared according to Example 23 and dialyzedagainst 20 mM HEPES-buffer pH 7.4 (containing 150 mM NaCl and 3%sucrose). Then the solution was diluted to a final concentration of 300μg/ml with 20 mM HEPES-buffer pH 7.4 (containing 150 mM NaCl and 3%sucrose). Subsequently mPEG succinimidyl succinate (chain length: 5 kD)was added (concentration: 25 mg PEG SS 5000/mg VWF) and thefurin-maturated VWF was PEGylated for 1 h at room temperature. Then thereagent was separated from the PEGylated VWF by UF/DF using a 100 kDmembrane consisting of regenerated cellulose (Millipore).

Example 25 In Vitro Characterization of the Furin-Maturated rVWF

The FVIII binding capacity of the furin-maturated rVWF was determined bysurface plasmon resonance technology and by ECA, as described in Example12 and FIG. 15, respectively. The results were compared to analbumin-free plasma-derived VWF preparations prepared according toExample 21.

As demonstrated in FIGS. 18 and 19, the FVIII binding-capacity of thefurin-maturated rVWF was comparable with a plasma-derived referencepreparation.

Example 26 Conjugation of VWF with Polysialic Acid by Cross-Linking withGlutaraldehyde

For the conjugation of rVWF with polysialic acid (colominic acid) usingglutaraldehyde as cross-linking reagent (Migneault et al., Biotechniques37: 790-796, 2004) 4 ml of a solution of colominic acid (concentration:20 mg/ml) in 20 mM HEPES-buffer (150 mM NaCl, pH 7.4) are prepared andthe pH is adjusted to 7.4 by addition of 0.1 M NaOH. Glutaraldehyde isadded to give a final concentration of 0.01%. Subsequently 1 ml of asolution of rVWF (400 μg/ml) in 20 mM HEPES-buffer (150 mM NaCl, pH 7.4)is added in aliquots of 100 μl and the mixture is incubated for 1 hunder gentle shaking. Then the mixture is dialyzed, trehalose is added(final concentration: 3%) and the rVWF-polysialic acid conjugate isconcentrated by ultrafiltration.

Example 27 Increase of VWF Half-Life in VWF-Deficient Mice

VWF-deficient mice described by Denis et al. (PNAS 95: 9524-9529, 1998)were used as an animal model of human VWD. Groups of 5 mice received abolus injection (10 ml/kg) via the tail vein with either rVWF-polysialicacid conjugate prepared according to Example 19 or native rVWF toachieve 100 U VWF:Ag/kg. Citrate plasma by heart puncture afteranesthesia was prepared from the respective groups, 5 min, 1, 3, 6, 9and 21 h after injection. VWF antigen recovery and endogenous mouseFVIII activity levels were measured in the plasma samples. The resultsof this experiment are summarized in FIGS. 21 and 22.

Half-life of VWF was calculated using the MicroMath Scientist program(Micromath Research, Saint Luis, Mo., US) employing one compartmentmodel from the pharmacokinetic library. Area under curve for FVIIIactivity was calculated by a trapezoidal model with baselinesubtraction.

Half-life of VWF increased from 1.3 h (native rVWF) to 2.4 h(rVWF-polysialic acid conjugate), AUC for FVIII increased from 3.3U*hr/ml to 5.3 U*hr/ml respectively.

Example 28 Coupling of mPEG Propionaldehyde to Lysine Groups byReductive Amination

A rVWF solution (400 μg/ml) is prepared in 0.05 M sodium phosphatebuffer, pH 7.2 and mPEG propionaldehyde (chain length 5 kDa) is added togive a final concentration of 10 mg mPEG propionaldehyde per mg VWF.This mixture is stirred for 30 minutes. Then NaCNBH₃ is added (1 mg/mgrVWF) and the mixture is incubated for 15 h at room temperature undergentle shaking. An aqueous 1 M TRIS-solution, pH 7.2 is added (50 μl permg NaCNBH₃) and stirred for 1 h to terminate the reaction. Subsequently,the PEGylated rVWF is separated from the reagents byultrafiltration/diafiltration using a 100 kD membrane (regeneratedcellulose/Millipore).

Example 29 N-terminal PEGylation of VWF

N-terminal PEGylation of rVWF is carried out as described by Lee et al.(Pharm. Res. 20: 818-825, 2003). A solution of rVWF (finalconcentration: 500 μg/ml) is prepared in 50 mM sodium acetate buffer, pH5.5 and mPEG propionaldehyde (chain length: 5 kD) is added(concentration: 10 mg mPEG propionaldehyde/mg VWF). The PEGylation iscarried out for 24 h at room temperature in the presence of 2 mM NaCNBH₃as reducing agent. Subsequently, the PEGylated rVWF is separated fromthe reagents by ultrafiltration/diafiltration using a 100 kD membrane(regenerated cellulose/Millipore).

Example 30 Sequential PEGylation of Lysine-Residues and SH-Residues ofrVWF

RVWF is PEGylated via lysine residues with mPEG succinimidyl succinate(chain length: 5 kD) according to Example 2. The PEGylation is performedat room temperature for 1 h and the free reagents are separated from therVWF PEG conjugate by UF/DF against 20 mM HEPES-buffer, pH 7.4,containing 5% saccharose using a 100 kD membrane (regeneratedcellulose/Millipore). Then the pH value of the solution is adjusted to7.6 with 0.1 M NaOH and mPEG maleimide (chain length 5 kD/finalconcentration 10 mM) is added for PEGylation of free SH-groups. ThePEGylation is carried out for 2 h at room temperature under gentleshaking. Then the reagents are separated again from the reaction mixtureusing a 100 kD membrane (regenerated cellulose/Millipore).

Example 31 Enzymatic Oxidation of Carbohydrate Residues and SubsequentPEGylation with PEG-Hz

The enzymatic oxidation of carbohydrate residues (Wilchek et Bayer,Meth. Enzymol. 138; 429-442, 1987) in rVWF to create aldehyde groups iscarried out as described by Avigad et al. (J. Biol. Chem. 237: 2736-43,1962) by use of galactose oxidase from Dactylium dendroides (Sigma). Thesolution obtained is dialyzed against 50 mM phosphate buffer, pH 7.2 anddiluted to a VWF concentration of 400 μg/ml. Than mPEG hydrazide (mPEGHz) with a chain length of 5 kD (final concentration: 40 mg mPEG-Hz/mgVWF) is added. The mixture is incubated at room temperature under gentleshaking for 3 h. The reagents are separated from the PEGylated rVWF byUF/DF against 20 mM HEPES-buffer (150 mM NaCl, pH 7.4) containing 5%saccharose using a 100 kD membrane (regenerated cellulose/Millipore).

Example 32 PEGylation of rVWF with Blocking of the FVIII Binding Site byrFVIII

For preparation of PEGylated rVWF with unmodified FVIII binding site 3ml of a solution containing rFVIII (200 U/ml) and rVWF (40 U VWF:Ag/ml)in 50 mM HEPES-buffer (50 mM HEPES, 150 mM NaCl, 2% trehalose, pH 7.4)are prepared and incubated for 1 h at 37° C. The mixture is cooled toroom temperature and PEG succinimidyl succinate (PEG-SS/chain length: 5kD) is added (final concentration: 1 mg PEG-SS/U VWF:Ag) and incubatedfor 1 h under gentle shaking. Then CaCl₂ is added under gentle shakingto give a final concentration of 400 mM. This solution is applied onto achromatographic column (2.6×80 cm) filled with Sephacryl S-400 HR(Amersham) and the PEGylated rVWF with a free binding site for FVIII isseparated from the rFVIII by size-exclusion chromatography (elutionbuffer: 50 mM HEPES-buffer, 400 mM CaCl₂, pH 7.4).

Example 33 PEGylation of rVWF with Blocking of the FVIII Binding Site byHeparin

5 ml of a solution of rVWF (300 μg/ml) in 50 mM HEPES-buffer, pH 7.4 areprepared and added to 2 ml of a suspension of Heparin-Sepharose CL-6B(Amersham Bioscience) in the same buffer. This mixture is incubated for2 h under gentle shaking and the VWF is bound to the gel (de Romeuf etMazurier, Thromb. Hamost. 69: 436-440, 1993). Subsequently mPEGsuccinimidyl succinate (200 mg/mg MT) is added to the mixture and thePEGylation is carried out at room temperature under gentle shaking for 1h. The mixture is diluted with an equal volume of HEPES-buffer, pH 7.4containing 2M NaCl. The gel is separated from the supernatant byfiltration. Then the gel is washed 3× with 2 ml HEPES-buffer, pH 7.4 (20mM HEPES, 1 M NaCl) and the supernatant and the washing solutions arecombined. Subsequently the solution containing the PEGylated VWF isconcentrated by ultrafiltration and diafiltrated against 20 mMHEPES-buffer, pH 7.4 (150 mM NaCl, 3% saccharose) using a 100 kDmembrane consisting of regenerated cellulose (Millipore). The derivativeobtained shows full FVIII binding capacity by use of the Biacoretechnology or the ECA test (the test systems are described in U.S.Provisional Patent Application Ser. No. 60/668,378 filed Apr. 4, 2005).

Example 34 Conjugation of VWF with Hyaluronic Acid

The modification of lysine residues with hyaluronic acid (HA) wascarried out by reductive amination using hyaluronic acid from Sigma (C53747). HA was dissolved in a freshly prepared 0.1 M NaIO₄ solution togive a final concentration of 5 mg HA/ml. Then the oxidation was carriedout for 15 min in the dark under gentle stirring. The reaction wasstopped by the addition of 2 ml ethylene glycol per ml oxidized HAsolution and further stirring for 30 min in the dark at roomtemperature. The solution was dialyzed over night against 0.05 M sodiumphosphate buffer, pH 7.2 in the dark at 4° C. Subsequently an aliquot ofthis solution was added to a rVWF solution (40 U VWF:Ag/ml) in 0.05 Msodium phosphate buffer, pH 7.2 to give a final concentration of 50 mgactivated HA per mg protein. This mixture was stirred for 120 min atroom temperature in the dark. NaCNBH₃ was added (1 mg/mg protein) andthe mixture was incubated for 18 h at room temperature in the dark undergentle shaking. Then 100 μl M Tris-buffer, pH 7.2 was added per ml ofthis mixture and stirred for 1 h to terminate the reaction. The freereagents were separated from the rVWF HA conjugate by UF/DF using a 100kD membrane (regenerated cellulose/Millipore).

Example 35 Biochemical In Vitro Characterization of PSA Conjugated VWF

RVWF was polysialylated according to Example 19. In addition to theparameters described in this example such as isoelectric focusing (FIG.20), the determination of FVIII binding capacity by the ECA-test (54%)according to Example 16 and by surface plasmon resonance (70%) accordingto Example 12 the ratio VWF:RCo/VWF:Ag was calculated. The VWF antigenlevel was determined by use a commercially available assay system(Asserachrom vWF, Roche, Basel, Switzerland). The functional activity ofthe preparation was determined by the Ristocetin Co-factor assay asdescribed by Macfarlane et al. (Thromb. Diath. Haemorrh 34:306-308,1975). The ratio of 0.39 calculated for the rVWF starting materialdecreases to 0.13 after polysialylation.

Example 36 Conjugation of rVWF with Branched Peg Via Lysine Residues

A solution of a mature rVWF (35 U VWF:Ag/ml) in 20 mM HEPES buffer, 150mM NaCl, pH 7.4, containing 0.5% sucrose was prepared according toExample 23. Then branched mPEG succinimidyl glutarate (PE-SG/chainlength: 20 kD) supplied by NOF corporation (NOF Europe, Grobbendonk,Belgium) was added to this solution under gentle stirring (5 mgPEG-SG/mg protein) and the pH value was adjusted to 7.4 by drop wiseaddition of 0.5 M NaOH. Than the PEGylation was carried out under gentlestirring for 1 h at room temperature. Subsequently the reaction mixturewas applied onto an equilibrated ion-exchange chromatography resin(Fractogel EMD TMAE 650M) in 20 mM HEPES buffer, 150 mM NaCl, pH 7.4,containing 0.5% sucrose. Then the column was washed with 20 CVequilibration buffer to remove excess reagent and the PEGylated rVWF waseluted with elution buffer (20 mM HEPES, 0.5 M NaCl, 0.5% sucrose, pH7.4). The eluate was concentrated by ultrafiltration/diafiltration witha membrane consisting of regenerated cellulose and with a molecularweight cut-off of 100 kD using a buffer system consisting of 20 mMHEPES, 150 mM NaCl, 0.5% sucrose, pH 7.4. The PEGylated derivativeobtained showed a slightly diminished VWF:Rco/VWF:Ag ratio of 0.79 ascompared with the rVWF starting material (VWF:Rco/VWF:Ag ratio: 0.89).In addition, the PEGylated rVWF starting material had a FVIII bindingcapacity of 83% as measured by the ECA test (Example 16).

Example 37 Pharmacokinetics of VWF Conjugated with Branched PEG's inFVIII-K.O.-Mice

FVIII deficient mice (Bi et al., Nat. Genet. 10: 119-121, 1995) wereused as a model of severe human hemophilia A. Groups of 5 mice receiveda bolus injection (10 ml/kg) via the tail vein with either a mixture ofPEG-rVWF (branched PEG, SG) and rFVIII or a mixture of native rVWF andrFVIII to achieve 30 U FVIII/ml and 25 U VWF/ml. Five min, 3, 9, 24 and32 h after injection citrate plasma was prepared from the respectivegroups by heart puncture after anesthesia. FVIII activity and VWFantigen recovery levels were measured in plasma samples. The eliminationcurves for VWF and FVIII are shown in FIG. 23 and FIG. 24. VWF half-lifeincreased from 1.4 to 9.7 h, AUC for VWF increased from 11.8 to 49.2U*h/ml. Half-life for FVIII increased from 1.2 h (in the presence ofnative rVWF) to 4.4 h (when applied together with PEG-rVWF), area undercurve (AUC) increased from 12.1 to 30.5 U*h/ml.

Example 38 Comparative Pharmacokinetics of FVIII Mixed with DifferentAmounts of PEG-VWF in FVIII-K.O.-Mice

Groups of 5 FVIII deficient K.O.-mice received a bolus injection (10ml/kg) via the tail vein with various mixtures of PEG-rVWF (PEGylationof lysine residues with 25 mg branched PEG-SG 20000/mg protein) andrFVIII (A: 20 IU PEG-rVWF/ml+20 IU FVIII/ml; B: 10 IU PEG-rVWF/ml+20 IUFVIII/ml; C: 3 IU PEG-rVWF/ml+20 IU FVIII/ml). For the PEGylated rVWF aratio of 3 mole PEG/mole lysine was calculated. Comparing the samequantities (based on units) of PEG-rVWF and rFVIII in the differentPEG-rVWF/rFVIII mixtures the following ratios of PEG/FVIII could becalculated: 3:1 (A); 1.5:1 (B): 0.45:1 (C). After anesthesia citrateplasma was prepared by heart puncture from the respective groups 5 min,1, 3, 9, and 24 h after injection. No relevant difference in FVIIIhalf-life (A: 2.1 h, B: 2.0 h, C, 2.5 h) and AUC (A: 18.9, B: 14.5 andC, 13.2 U*hr/ml) was found. The elimination curves for FVIII are shownin FIG. 25.

Example 39 Comparative Pharmacokinetics Using PEG-VWF Preparations withDifferent Degrees of PEGylation

FVIII×VWF double knockout mice were obtained by cross-breeding of FVIIIdeficient and VWF-deficient mice. Those mice suffer from FVIIIdeficiency as well as from VWF deficiency. Groups of 5 FVIII×VWF doubleknockout mice were infused via the tail vein with a mixture of nativerVWF/rFVIII (100/150 IU/kg) or with PEGylated rVWF #A mixed with rFVIII(100/150 IU/kg) or with PEGylated rVWF #6 mixed with rFVIII (150/150IU/kg). PEG-rVWF #A (5 mg PEG-SS 5000/mg protein) and PEG-rVWF #6 (20 mgPEG-SS 5000/mg protein) were prepared according to Example 24. Forpreparation #A a ratio of 2.5 mole PEG/mole lysine and for preparation#B a ratio of 10 mole PEG/mole lysine was calculated. For this citrateplasma samples were prepared 5 min, 1, 3, 9 and 24 h after sampleapplication. Plasma levels of VWF:Ag and FVIII activity were measuredand expressed as percent of the maximum plasma level, generally reached5 min after injection. The elimination curves for VWF and FVIII areshown in FIG. 26 and FIG. 27, respectively. Half-life was 6.3 h and 8.1h for PEG-rVWF #A and #6 respectively. For the native rVWF a half-lifeof 2.0 h was calculated. The normalized AUC (% of maximum×h) for bothPEGylated rVWFs increased from 360%*h (native rVWF) to 901%*h (#A) and1064%*h (#6). The circulation time for co-infused rFVIII was improved byPEGylated rVWF compared with native VWF. The half-life of FVIII was 0.8h in the presence of native rVWF, and increased to 1.5 and 1.8 h wheninfused with PEGylated rVWF #A and #13 respectively. The AUC for FVIIIwas 214, 370 and 358%*h.

Example 40 PEGylation of VWF-Dimer

A solution of a VWF dimer (58 IU VWF:Ag/ml), which was purified from theconditioned medium of a recombinant CHO cell line (Baxter BioScience),was prepared in 20 mM HEPES buffer, 150 mM NaCl, pH 7.4, containing 0.5%sucrose was prepared. Then branched mPEG succinimidyl glutarate(PE-SG/chain length: 20 kD) supplied by NOF corporation was added tothis solution under gentle stirring (5 mg PEG-SG/mg protein) and the pHvalue was adjusted to 7.4 by drop wise addition of 0.5 M NaOH. ThePEGylation was carried out under gentle stirring for 1 h at roomtemperature. Subsequently the reaction mixture was applied onto anequilibrated ion-exchange chromatography resin (Fractogel EMD TMAE 650M)in 20 mM HEPES buffer, 150 mM NaCl, pH 7.4, containing 0.5% sucrose.Then the column was washed with 20 CV equilibration buffer to removeexcess reagent and the PEGylated rVWF dimer was eluted with elutionbuffer (20 mM HEPES, 0.5 M NaCl, 0.5% sucrose, pH 7.4). The eluate wasconcentrated by ultrafiltration/diafiltration with a membrane consistingof regenerated cellulose (Millipore) and with a molecular weight cut-offof 100 kD using a buffer system consisting of 20 mM HEPES, 150 mM NaCl,0.5% sucrose, pH 7.4.

Example 41 PEGylation and In Vitro Characterization of Low Multimer rVWF

Mature rVWF was purified according to Example 23. The purificationprocedure included ion-exchange chromatography steps as well as a finalgelfiltration step on Superose 6, performed in 20 mM HEPES, 150 mM NaCl,pH 7.4, where the high multimer rVWF (17 multimers) was eluted in thevoid volume. The VWF multimer analysis was performed according toRuggeri and Zimmerman (Blood 57: 1140-43, 1981) using a 1.0% agarosegel. A low multimer rVWF preparation (6 multimers) was obtained from aside fraction, eluting at higher retention times. This fraction wasstabilized by addition of 0.5% sucrose, pH 7.4. Then the low multimerrVWF was PEGylated using mPEG succinimidyl succinate (PEG-SS). ThePEG-SS was added to this solution under gentle stirring (5 mg PEG-SS/mgprotein) and the pH value was adjusted to 7.4 by drop wise addition of0.5 M NaOH. The PEGylation was carried out under gentle stirring for 1 hat room temperature. Subsequently the excess reagent was removed byultrafiltration/diafiltration with a membrane consisting of regeneratedcellulose and with a molecular weight cut-off of 100 kD using a buffersystem consisting of 20 mM HEPES, 150 mM NaCl, 0.5% sucrose, pH 7.4. TheFVIII binding capacity determined by the ECA assay according to Example16 slightly decreased from 49% for the starting material to 34% for thePEGylated preparation. The VWF:RCoNWF:Ag ratio of 0.02 measured for thelow multimer rVWF was not affected by the PEGylation procedure.

Example 42 Derivatization of VWF with Reversibly Blocked FVIII BindingEpitopes (Blocking with FVIII and Heparin)

A chromatographic column (15 mm×148 mm) was filled with Heparin HyperD(Bio-Sepra) and equilibrated with an equilibration buffer consisting of20 mM HEPES, 68 mM NaCl, 0.5% sucrose, pH 7.4. Then a solution of maturerVWF (48 IU VWF:Ag/ml) in 20 mM HEPES, 150 mM NaCl, 0.5 sucrose wasdiluted with H₂O to give a conductivity of 7-8 mS/cm and applied ontothis column using a linear flow rate of 1.5 cm/min. Subsequentlybranched mPEG succinimidyl glutarate (chain length: 20 kD) supplied byNOF corporation (NOF Europe, Grobbendonk. Belgium) was freshly dissolvedin 15 ml equilibration buffer to give a final concentration of 5 mgPEG-SG/mg bound protein. Then this reagent solution was pumped onto thecolumn and the PEGylation was carried out for 2 hours under staticconditions. Then the column was washed with 10 CV equilibration buffercontaining 0.05% lysine. Then the PEGylated rVWF with protected FVIIIbinding epitope was eluted with a buffer consisting of 20 mM HEPES, 1 MNaCl, 0.5% sucrose, pH 7.4. Finally this solution was concentrated byultrafiltration/diafiltration against 20 mM HEPES-buffer, pH 7.4 (150 mMNaCl, 0.5% sucrose) using a 100 kD membrane consisting of regeneratedcellulose (Millipore). The derivative obtained showed a VWF:RCoNWF:Agratio of 0.48, which was identical to the rVWF starting material (ratio0.47). In contrast to the PEGylation procedure of rVWF with branchedPEG-SG 20000 as described in Example 36 the FVIII binding capacity wasnot affected by this PEGylation procedure with FVIII epitope capping asmeasure by the ECA test (Example 16).

Example 43 Conjugation of VWF with Degradable PEG Via Lysine Residues

A mature rVWF is purified according to Example 23. Then a solution ofthis rVWF (40 U VWF:Ag/ml) in 20 mM HEPES buffer, 150 mM NaCl, pH 7.4,containing 0.5% sucrose is prepared. Subsequently the PEGylation iscarried out by adding mPEG-(carboxymethyl)-3-hydroxy-butanoic acidN-hydroxysuccinimide ester (chain length: 5 kD) to this solution undergentle stirring (5 mg PEG-reagent/mg protein) and the pH value isadjusted to 7.4 by drop wise addition of 0.5 M NaOH. Then the PEGylationreaction is carried out under gentle stirring for 1 h at roomtemperature. Subsequently the excess reagent is separated from thePEGylated rVWF by ultrafiltration/diafiltration with a membraneconsisting of regenerated cellulose and with a molecular weight cut-offof 100 kD using a buffer system consisting of 20 mM HEPES, 150 mM NaCl,0.5% sucrose, pH 7.4.

1. A method for forming a proteinaceous molecule comprising a vonWillebrand Factor (VWF) molecule and a PEG moiety covalently bound to atleast one carbohydrate residue on said VWF molecule, said proteinaceousmolecule having the capability of binding at least one Factor VIII(FVIII) molecule, comprising: a) providing a VWF molecule selected fromthe group consisting of plasmatic VWF, recombinant VWF, and dimers andmultimers thereof; b) oxidizing carbohydrate residues on said VWFmolecule; c) contacting said carbohydrate residues with a PEG reagentcontaining a hydrazide group; and d) allowing said PEG hydrazide reagentto covalently bind to at least one carbohydrate residue on said VWF,thereby forming said proteinaceous molecule, wherein the in vivohalf-life of said proteinaceous molecule is increased as compared to thein vivo half-life of a VWF molecule and the in vivo half-life of saidFVIII molecule bound to said proteinaceous molecule is increased ascompared to the in vivo half-life of a FVIII molecule not bound to saidproteinaceous molecule.
 2. The method of claim 1 wherein said oxidizingstep of (b) is conducted using a chemical oxidizing agent.
 3. The methodof claim 2 wherein said chemical oxidizing agent is NaIO₄.
 4. The methodof claim 1 wherein said oxidizing step of (b) is conducted using anenzymatic oxidizing agent.
 5. The method of claim 4 wherein saidenzymatic oxidizing agent is galactose oxidase.
 6. The method of claim 1wherein said PEG reagent is selected from the group consisting of linearalkoxy PEG, linear bifunctional PEG, branched PEG, multi-armed PEG,forked PEG, PEG attached to a polyol core, dendritic PEG, PEG withstable linkages, PEG with degradable linkages, and PEG with hydrolysablelinkages.
 7. The method of claim 6 wherein said PEG reagent has aninternal structure selected from the group consisting of homopolymer,alternating copolymer, random copolymer, block copolymer, alternatingtripolymer, random tripolymer, and block tripolymer.
 8. The method ofclaim 1 whereby said PEG reagent is provided at a ratio of about 0.1 toabout 200 mg PEG/mg VWF protein.
 9. The method of claim 1 whereby saidPEG reagent is provided at a ratio of about 1.0 to about 25 mg PEG/mgVWF protein.
 10. A method for forming a proteinaceous construct moleculecomprising a von Willebrand Factor (VWF) molecule and a polysialic acid(PSA) moiety covalently bound to at least one lysine residue on said VWFmolecule, said proteinaceous molecule having the capability of bindingat least one Factor VIII (FVIII) molecule, comprising: a) providing aVWF molecule selected from the group consisting of plasmatic VWF,recombinant VWF, and dimers and multimers thereof; b) contacting saidVWF molecule with a PSA reagent containing an aldehyde; c) allowing saidPSA reagent to covalently bind to at least one lysine residue on saidVWF to form a Schiff base in solution; and d) contacting said solutionwith a reducing agent to form a secondary amino bond; thereby formingsaid proteinaceous molecule, wherein the in vivo half-life of saidproteinaceous molecule is increased as compared to the in vivo half-lifeof a VWF molecule and the in vivo half-life of said FVIII molecule boundto said proteinaceous molecule is increased as compared to the in vivohalf-life of a FVIII molecule not bound to said proteinaceous molecule.11. The method of claim 10 wherein said reducing agent is NaCNBH₃.
 12. Amethod for forming a proteinaceous construct molecule comprising a vonWillebrand Factor (VWF) molecule and a hyaluronic acid (HA) moietycovalently bound to at least one lysine residue on said VWF molecule,said proteinaceous molecule having the capability of binding at leastone Factor VIII (FVIII) molecule, comprising: a) providing a VWFmolecule selected from the group consisting of plasmatic VWF,recombinant VWF, and dimers and multimers thereof; b) contacting asolution of HA with an oxidizing agent to form activated HA; c)contacting said VWF molecule with said activated HA, thereby formingsaid proteinaceous molecule, wherein the in vivo half-life of saidproteinaceous molecule is increased as compared to the in vivo half-lifeof a VWF molecule and the in vivo half-life of said FVIII molecule boundto said proteinaceous molecule is increased as compared to the in vivohalf-life of a FVIII molecule not bound to said proteinaceous molecule.13. The method of claim 1 wherein the step of oxidizing carbohydrateresidues on said VWF molecule comprises contacting a carbohydrateresidue on said VWF molecule with an oxidizing enzyme to form anoxidized carbohydrate moiety on said VWF.